泛素化介导的非蛋白质降解功能

泛素因标记被26 S蛋白酶体降解的蛋白质而著名．然而近几年发现,泛素作用远不止此,不仅具有参与蛋白质降解这一重要“传统作用”,还起着比先前想象更多变的、更精美的细胞调控作用,是非常重要的细胞过程的多层面调节因子,具有许多重要的非蛋白质降解功能,包括DNA损伤修复、DNA复制、信号传导、转录调节、膜运输、胞吞、蛋白激酶活化、染色质重塑和病毒芽殖．这些功能涉及多聚泛素化和单泛素化及多泛素化．因此,泛素化异常可能涉及疾病的发生和发展．对这些功能的了解可以拓展人们对泛素的认识,有助于对多种细胞过程的深入理解,也有助于相关新药的研发．     

Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.     

A SIM-ultaneous role for SUMO and ubiquitin

Ubiquitin and ubiquitin-like proteins (Ubls) share a beta-GRASP fold and have key roles in cellular growth and suppression of genome instability. Despite their common fold, SUMO and ubiquitin are classically portrayed as distinct, and they can have antagonistic roles. Recently, a new family of proteins, the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligases (STUbLs), which directly connect sumoylation and ubiquitylation, has been discovered. Uniquely, STUbLs use SUMO-interaction motifs (SIMs) to recognize their sumoylated targets. STUbLs are global regulators of protein sumoylation levels, and cells lacking STUbLs display genomic instability and hypersensitivity to genotoxic stress. The human STUbL, RNF4, is implicated in several diseases including cancer, highlighting the importance of characterizing the cellular functions of STUbLs.     

Ubiquitin-based anticancer therapy: Carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors

The proteasome inhibitor bortezomib remains the only ubiquitin pathway effector to become a drug (VELCADE?) and has become a successful treatment for hematological malignancies. While producing a global cellular effect, proteasome inhibitors have not triggered the catastrophe articulated initially in terms such as "buildup of cellular garbage". Proteasome inhibitors, in fact, do have a therapeutic window, although in the case of the prototype bortezomib it is small owing to peripheral neuropathy, myelosuppression and, as recently reported, cardiotoxicity [1]. Currently, several second-generation molecules are undergoing clinical evaluation to increase this window. An alternative strategy is to target ubiquitin pathway enzymes acting at non-proteasomal sites-E1, E2, and E3, associated with ubiquitin conjugation, and deubiquitylating enzymes ("DUBs")-that act locally on selected targets rather than on the whole cell. Inhibitors (or activators, in some cases) of these enzymes should be developable as selective antitumor agents with toxicity profiles superior to that of bortezomib. Various therapeutic hypotheses follow from known cellular mechanisms of these target enzymes; most hypotheses relate to cancer, reminiscent of the FDA-approved protein kinase inhibitors now marketed. Since ubiquitin tagging controls the cellular content, activity, or compartmentation of proteins associated with disease, inhibitors or activators of ubiquitin conjugation or deconjugation are predicted to have an impact on disease. For practical and empirical reasons, inhibitors of ubiquitin pathway enzymes have been the favored therapeutic avenue. In approximately the time that has elapsed since the approval of bortezomib in 2003, there has been some progress in developing potential anticancer drugs that target various ubiquitin pathway enzymes. An E1 inhibitor and inhibitors of E3 are now in clinical trial, with some objective responses reported. Appropriate assays and/or rational design may uncover improved inhibitors of these enzymes, as well as E2 and DUBs, for further development. Presently, it should become clear whether one or both of the two general strategies for ubiquitin-based drug discovery will lead to truly superior new medicines for cancer and other diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Proteome-wide identification of ubiquitylation sites by conjugation of engineered lysine-less ubiquitin

Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27(Kip1). Mutation of this site thus inhibited ubiquitylation of and stabilized p27(Kip1), suggesting that this lysine residue is the target site of p27(Kip1) for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.     

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7^ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7^ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7^ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7^ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7^ADR cells. p53 ubiquitylation is markedly increased in MCF7^ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7^ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Regulatory functions of ubiquitin in diverse DNA damage responses

In recent years there has been intense investigation and rapid progress in our understanding of the cellular responses to various types of endogenous and exogenous DNA damage that ensure genetic stability. These studies have identified numerous roles for ubiquitylation, the post-translational modification of proteins with single ubiquitin or poly-ubiquitin chains. Initially discovered for its role in targeting proteins for degradation in the proteasome, ubiquitylation functions in a variety of regulatory roles to co-ordinate the recruitment and activity of a large number of protein complexes required for recovery from DNA damage. This includes the identification of essential DNA damage response genes that encode proteins directly involved in the ubiquitylation process itself, proteins that are targets for ubiquitylation, proteins that contain ubiquitin binding domains, as well as proteins involved in the de-ubiquitylation process. This review will focus on the regulatory functions of ubiquitylation in three distinct DNA damage responses that involve ubiquitin modification of proliferating cell nuclear antigen (PCNA) in DNA damage tolerance, the core histone H2A and its variant H2AX in double strand break repair (DSBR) and the Fanconi anaemia (FA) proteins FANCD2 and FANCI in cross link repair.     

A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2

Background  

Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.     

USP7: Structure,substrate specificity,and inhibition

Turnover of cellular proteins is regulated by Ubiquitin Proteasome System (UPS). Components of this pathway, including the proteasome, ubiquitinating enzymes and deubiquitinating enzymes, are highly specialized and tightly regulated. In this mini-review we focus on the de-ubiquitinating enzyme USP7, and summarize latest advances in understanding its structure, substrate specificity and relevance to human cancers. There is increasing interest in UPS components as targets for cancer therapy and here we also overview the recent progress in the development of small molecule inhibitors that target USP7.     

A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.     

Structure of S5a bound to monoubiquitin provides a model for polyubiquitin recognition

Ubiquitin is a key regulatory molecule in diverse cellular events. How cells determine the outcome of ubiquitylation remains unclear; however, a likely determinant is the specificity of ubiquitin receptor proteins for polyubiquitin chains of certain length and linkage. Proteasome subunit S5a contains two ubiquitin-interacting motifs (UIMs) through which it recruits ubiquitylated substrates to the proteasome for their degradation. Here, we report the structure of S5a (196-306) alone and complexed with two monoubiquitin molecules. This construct contains the two UIMs of S5a and we reveal their different ubiquitin-binding mechanisms and provide a rationale for their unique specificities for different ubiquitin-like domains. Furthermore, we provide direct evidence that S5a (196-306) binds either K63-linked or K48-linked polyubiquitin, and in both cases prefers longer chains. On the basis of these results we present a model for how S5a and other ubiquitin-binding proteins recognize polyubiquitin.     

Ubiquitin in homeostasis,development and disease

Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.     

Ubiquitin Signaling Regulates RNA Biogenesis,Processing, and Metabolism

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.     

The ubiquitin-proteasome system

The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first ‘tagged’ by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.     

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins

The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Identification of ubiquitin target proteins using cell-based arrays

A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significance of ubiquitination. There are, however, a few effective screening methods for rapid analysis of ubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combined with cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulate to form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consisted of 112 cDNAs encoding key components of major cellular pathways and potential targets of polyubiquitination. Among them, 40 proteins formed accumulation dots in response to proteasome inhibitor, MG-132, treatment. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK, and RhoGDI2, are previously not known as the targets of ubiquitin. We further validated our findings by examining the endogenous counterparts of some of these proteins and found that those endogenous proteins form a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.     

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus–host protein–pro-tein interaction networks can reveal cellular pathways critical to viral replication and disease patho-genesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as crit-ical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for fur-ther studies of flavivirus–host interactions, disease pathogenesis, and new drug targets.